The early onset of Prox1 expression is revealed by β-galactosidase expression and in situ hybridization.

<p>Whole mount β-galactosidase histochemcial reaction using X-Gal was performed in <i>Prox1</i> heterozygous and nullizygous embryos. A. Starting at E11.0, a progressive upregulation of <i>Prox1</i> is seen in the anterior (AC) and posterior (PC) canal cristae. B. By E13.5, expression is also detected in the horizontal canal crista (HC), the striolar region of the utricle (U), the canals and the endolymphatic duct (ED); expression in the saccule is barely detected (S). In the cochlea, upregulation of β-galactosidase expression is detected in the apex and decreases toward the base. Arrows indicate expression in anterior and posterior canal with their expression. C. Expression of β-galactosidase is identical in heterozygous and nullizygous mice with the exception that the signal is stronger in nullizygous mice. Faint β-galactosidase expression is also detected in the delaminating spiral ganglion neurons (SPG; C and insert in B,C). D. In situ hybridization shows at E14.5 expression in the canal cristae and the cochlea, but indicates a more prominent upregulation in the base at this stage. Only spiral ganglion sensory neurons are faintly positive for <i>Prox1</i> in situ (SPG in D). E,F At postnatal stages, <i>Prox1</i> expression remains in the canal cristae as revealed by in situ hybridization for <i>Prox1</i> mRNA or X-Gal reaction, but does not show the extensive expression in the non-sensory parts of the canals as in earlier stages (insert in F). Bar, 100 µm.</p

Immunolabeling and dye tracing reveals Type II fiber disorganization in the organ of Corti of Prox1^flox/flox; Tg(Nes-Cre) conditional mutant embryos.

<p>(A, A′,A″) This 1 day old basal turn shows that the Prox1 protein is present in the supporting cells (A′, A″) and that neuron processes extend beyond the first row of Deiter's cells to form a bundle of intertwined fibers near the second and third row of Prox1 positive Deiter's cells. Inserts in A, A′ and A″ show tubulin immunostaining in spiral ganglion cells (SPG) but show no immunoreaction for Prox1. The disorganization of nerve fibers becomes particularly obvious in a side by side comparison with the regular pattern of cells (shown with Hoechst stain) Type II process in wildtype (B,C). In the apex, Type II fibers extend in a random way towards base and apex between Prox1 positive supporting cells (D). Epoxy section of Prox1 (red) and tubulin immunostained (green) and Hoechst counterstained (blue) organ of Corti shows the normal organization of the greater epithelial ridge (GER) with Prox1 being restricted to 5 rows of supporting cells. Point applications of lipophilic dyes allows imaging the growth cones and their regular turns toward the base in wildtype (F,G) but shows a disorganized outgrowth and growth cones (GC) in <i>Prox1^flox/flox</i>; <i>Nes-Cre</i> conditional mutant mice. D1–3, Deiter's cells row 1–3; IP, inner pillar; OP, outer pillar; SPG, spiral ganglion. Bar, 100 µm (A–D), 50 µm (E–H; inserts in A,A′, A″).</p

Triple immunolabeling reveals cellular and fiber disorganization in the organ of Corti of Prox1^flox/flox; Tg(Pax2-Cre) conditional mutant embryos.

<p>Whole mount antibody staining of the organ of Corti highlighting the hair cells (anti-BDNF, blue), supporting cells (anti-Sox2, green) and nerve fibers (anti-β-tubulin, red). (A–C) The top row shows all three immunostaining together, the middle shows nerve fibers and supporting cells, and the bottom one nerve fibers and the hair cells. In contrast to the wild-type condition (A, A′ A″), in <i>Pax2-Cre;Prox1^flox/flox</i> conditional mutant embryos fibers extend beyond the first row of Deiter's cells (B′,C′) where they turn randomly toward the base or apex. In addition, hair cells are not in close proximity to the nerve fibers (A″, B″, C″). D1–D3− Deiter's cells, IP-inner Pillar cell, OP-outer Pillar cell, IHC-Inner hair cell, OHC1–3-outer hair cells. Bar, 100 µm.</p

Prox1 inactivation reduces the size of the anterior crista.

<p>As measured at E14.5, the length of the anterior cristae (AC) of <i>Prox1</i> mutant embryos is 20% reduced when compare vs. that of wild-type littermates. The size reduction is 30% when compared with the size of E18.5 <i>Prox1^flox/flox; Tg(Pax2-Cre)</i> mutant embryos. No significant changes in the length of the utricle were observed. Asterisks indicate a level of significance (p<0.05; <i>t-test</i>).</p

Effects of Prox1 loss-of function in the vestibular epithelia.

<p>A. X-gal staining of E14.5 <i>Prox1</i> heterozygous embryos reveals β-galactosidase activity in the anterior (AC) and horizontal (HC) parts of the canal cristae. B. Although morphologically normal, a reduction in the size of the crista epithelia is detected of <i>Prox1</i>-null littermates (white bar in the AC); gravistatic sensors such as utricle (U) show only transient <i>Prox1</i> expression and no apparent reduction in size. C, E. Hair cells are revealed using antibodies against Myo VII in a normal E18.5 <i>Prox1^flox/flox</i> conditional embryo. Note absence of imunoreactivity in the cruciate eminence (CE) of the anterior canal crista. E′. As shown by 2-photon activation, at this later stage, <i>Prox1</i> expression is high in supporting cells, but is also found in hair cells of the canal cristae as well as outside the sensory epithelium. Dotted line in B indicate the plane of sections through the horizontal canal crista, white arrows align lateral walls of the whole mount with the section. E,F. Despite the overlap of some Prox1 expression with hair cells in the canal cristae there is no morphologically obvious defects in hair cell differentiation other than reduced intensity of Myo VII staining are observed in <i>Prox1^flox/flox; Tg(Pax2-Cre)</i> as compared to <i>Prox1^flox/flox</i> littermates. However the reduction in size of the anterior canal crista (AC) is becoming more obvious at this late stage (C–F). CE-Cruciate eminence. Bar, 100 µm.</p

Organization of hair cells and supporting cells is mildly disrupted in Prox1^flox/flox;Pax2-Cre conditional mutant embryos.

<p>This image shows near radial thick (A,B) and ultrathin (C–F) sections through the middle turn of a <i>Prox1^flox/flox</i> control and a <i>Prox1^flox/flox</i>; <i>Pax2-Cre</i> conditional mutant animal. Note that the overall organization into 4 rows of hair cells (one inner and three outer) and five rows of supporting cells surrounding outer hair cells (two rows of pillar and three rows of Deiter's cells) is preserved in the conditional mutant (B,D,F). However, closer examination reveals that the regular organization of hair cells and supporting cells with two heads of pillar cells between inner and first row of outer hair cells (A,C,E) is only partially conserved in conditional mutants. In fact occasionally only a single pillar cell is found between inner and outer hair cells that appears to be the outer pillar cell (D,F). Hair cells develop normal with respect to apical kinocilia and stereocilia polarity and development (arrows in E,F). Abbreviations: D1–D3, first to third row of Deiter's cells; IHC, inner hair cell; IP, inner pillar cell; OHC, outer hair cell; OP, outer pillar cell. Bar indicates 100 µm in A,B and 10 µm in D–F).</p

Hair cell differentiation is not required for Prox1 expression.

<p>A, B. Prox1 expression is maintained in undifferentiated supporting cells of E18.5 <i>Atoh1</i>- null embryos. This result argues that <i>Prox1</i> expression is independent of hair cell mediated differentiation of sensory epithelia. A′ shows the Prox1 immunostaining in the apex. Abbreviations: AC, anterior crista; HC, horizontal crista; U, utricle. Bar, 100 µm.</p

Dye tracing reveals Type II fiber outgrowth problems in the organ of Corti of Prox1^flox/flox; Tg(Nes-Cre) conditional mutant embryos.

<p>NV Maroon (green) was inserted into the cochlear nucleus and NV Orange (red) was inserted into the olivocochlear bundle to label a small population of afferents (green) and all efferents (red). Efferents show a similarly organized intraganglionic spiral bundles in wildtype (A–C) and <i>Prox1^flox/flox</i>; <i>Nes-Cre</i> conditional mutant mice (D–J) and grow toether with afferents in radial fiber bundles (RF) to the organ of Corti. Note that at this stage only occasional efferents extent to outer hair cells. In contrast, type II afferents grow to the second or third row of outer hair cells (OHC) where they invariably turn toward the base (B,C). At this stage, none of the multiple type II afferents of <i>Prox1^flox/flox</i>; <i>Nes-Cre</i> conditional mutant mice show this coordinated growth pattern. Instead, fibers grow randomly toward the base or apex but mostly seem to stall with multiple branches extending toward the base and the apex (F,G). IGSB, intraganglionic spiral bundle; OHC, outer hair cells; RF, radial fibers; SPG, spiral ganglion. Bar, 100 µm (A–D), 50 µm (E–H; inserts in A,A′, A″).</p

A working hypothesis shows the differential expression of <i>Atoh1</i> in inner hair cells (IHC) and outer hair cells (OHC).

<p>Based on our <i>in situ</i> hybridization data, <i>Atoh1</i> is expressed earlier and at a higher level in IHCs (red line) than OHCs (green line), first in the base (top) followed by the apex (bottom). At early postnatal stages, <i>Atoh1</i> is downregulated to below the detection threshold of <i>in situ</i> hybridization, but a low level of <i>Atoh1</i> expression (grey lines) likely is sustained and can be detected by <i>Atoh1-LacZ</i> as late as P275 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030358#pone.0030358-Matei1" target="_blank">[14]</a>. In the CKO mutant cochlea, the <i>Atoh1</i> expression is reduced to a range of different levels shortly after its upregulation and is then eliminated after a few days (dashed lines). Some cells at the apex express <i>Atoh1</i> at a level close to control hair cells and can survive to adult stage.</p

A Novel Atoh1 “Self-Terminating” Mouse Model Reveals the Necessity of Proper Atoh1 Level and Duration for Hair Cell Differentiation and Viability

<div><p>Atonal homolog1 (<em>Atoh1</em>) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of <em>Atoh1</em> at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of <em>Atoh1</em> expression required for proper hair cell differentiation and maintenance remain unknown. We generated an <em>Atoh1</em> conditional knockout (CKO) mouse line using <em>Tg(Atoh1-cre)</em>, in which the <em>cre</em> expression is driven by an <em>Atoh1</em> enhancer element that is regulated by Atoh1 protein to “self-terminate” its expression. The mutant mice show transient, limited expression of <em>Atoh1</em> in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of <em>Atoh1</em> result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of <em>Atoh1, Barhl1</em> and <em>Pou4f3</em>, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that <em>Atoh1</em> is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that <em>Atoh1</em> expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered <em>Atoh1</em> CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with <em>Atoh1</em> therapy.</p> </div